1. Field of the Invention
Methods to obtain recombinant human erythropoietin (EPO) characterized by a sequence of tandem separation steps that includes differential precipitation, hydrophobic interaction, anionic exchange, cationic exchange and molecular exclusion liquid chromatographies. The EPO obtained by using the methods thus described.
2. Background Information
EPO is a glycoprotein that stimulates erythroblast differentiation in the bone marrow, thus increasing the circulating blood erythrocyte count. The mean life of erythrocytes in humans is 120 days and therefore, a human being losses 1/120 erythrocytes each day. This loss must be continuously restored to maintain a stable level of red blood cells.
The existence of EPO was first postulated by the turn of the century and was definitely proved by Reissman and Erslev early in the '50s. See Carnot, et al., C.R. Acad. Sci. (France), 143, 384–6 (1906); Carnot, et al., C.R. Acad. Sci. (France), 143, 432–5 (1906); Carnot, et al., C.R. Soc. Biol., 111, 344–6 (1906); Carnot, C.R. Soc. Biol., 111, 463–5 (1906); Reissman, Blood, 1950, 5, 372–80 (1950) and Erslev, Blood, 8, 349–57 (1953). Reissman and Erslev's experiments were promptly confirmed by other researchers. See Hodgson, et al., Blood, 9, 299–309 (1954); Gordon, et al., Proc. Soc. Exp. Biol. Med., 86, 255–8 (1954) and Borsook, et al., Blood, 9, 734–42 (1954).
The identification of the EPO production site in the organism was an issue of debate. Successive experiments led to identify the kidney as the main organ and peritubular interstitial cells as the synthesis site. See Jacobson, et al., Nature, 179, 633–4 (1957); Kuratowska, et al., Blood, 18, 527–34 (1961); Fisher, Acta Hematol., 26, 224–32 (1961); Fisher, et al., Nature, 205, 611–2 (1965); Frenkel, et al., Ann. N.Y. Acad. Sci., 149, 1, 292–3 (1968); Busuttil, et al., Proc. Soc. Exp. Biol. Med., 1137, 1, 327–30 (1971); Busuttil, Acta Haematol., (Switzerland), 47, 4, 238–42 (1972); Erslev, Blood, 44, 1, 77–85 (1974); Kazal, Ann. Clin. Lab. Sci., 5, 2, 98–109 (1975); Sherwood, et al., Endocrinology, 99, 2, 504–10 (1976); Fisher, Ann. Rev. Pharmacol. Toxicol., 28, 101–22 (1988); Jelkrnann, et al., Exp. Hematol., 11, 7, 581–8 (1983); Kurtz, et al., Proc. Natl. Acad. Sci. (USA), 80, 13, 4008–11 (1983); Caro, et al., J. Lab. Clin. Med., 103, 6, 922–31 (1984); Caro, et al., Exp. Hematol., 12, 357 (1984); Schuster, et al., Blood, 70, 1, 316–8 (1986); Bondurant, et al., Mol. Cell. Biol., 6, 7, 2731–3 (1986); Bondurant, et al., Mol. Cell. Biol., 6, 7, 2731–3 (1986); Schuster, et al., Blood, 71, 2, 524–7 (1988); Koury, et al., Blood, 71, 2, 524–7 (1988); Lacombe, et al., J. Clin. Invest., 81, 2, 620–3 (1988); Koury, et al., Blood, 74, 2, 645–51 (1989).
A smaller proportion, ranging from 10% to 15% of total EPO, is produced by the liver in adults. See Naughton, et al., J. Surg. Oncol., 12, 3, 227–42 (1979); Liu, et al., J. Surg. Oncol., 15, 2, 121–32 (1980); Dornfest, et al., Ann. Clin. Lab. Sci., 11, 1, 37–46 (1981); Dinkelaar, et al., Exp. Hematol., 9, 7, 796–803 (1981); Caro, et al., Am. J. Physiol., 244, 5 (1983); Dornfest, et al., J. Lab. Clin. Med, 102, 2, 274–85 (1983); Naughton, et al., Ann. Clin. Lab. Sci., 13, 5, 432–8 (1983); Jacobs, et al., Nature, 313, 6005, 806–10 (1985); Erslev, et al., Med. Oncol. Tumor. Pharmacother., 3, 3–4, 159–64 (1986). The EPO produced is directly proportional to the extent of tissular hypoxia and its expression rises by increasing the number of the EPO producing cells.
EPO has shown great efficiency in the treatment of anemia, especially anemia derived from renal failure. See Eschbach, et al., N. England J. of Med., 316, 2, 73–78 (1987); Krane, Henry Ford Hosp. Med J., 31, 3, 177–181 (1983). Its therapeutical usefulness, however, has been limited due to the unavailability of a massive production method. The quantity and quality of the EPO obtained by the extractive systems known were insufficient. Recently, the use of recombinant DNA technology has made it possible to obtain large amounts of proteins. The application of these techniques to eukaryotic cells has allowed a large scale production of EPO. See U.S. Pat. No. 5,688,679 (to Powell), U.S. Pat. No. 5,547,933 (to Lin), U.S. Pat. No. 5,756,349 (to Lin), U.S. Pat. No. 4,703,008 (to Lin) and U.S. Pat. No. 4,677,195 (to Hewick et al.).
Several techniques for the separation of glycoproteins such as EPO are currently available. Ultrafiltration, column electrofocusing, flat-bed electrofocusing, gel filtration, electrophoresis and isotachophoresis and some others chromatographic methods have been utilized for the purification of glycoproteins. The most widely used chromatographic techniques have been ionic exchange chromatography and adsorption chromatography.
The ionic exchange method is a separation technique by which the components of a solution are distinguished according to their different net charges and isolated by elution, either in stages or through the application of a continual gradient, with eluents of different ionic strength or pH. This method employs a gel or resin matrix, either of positive or negative charge, to induce binding or electrostatic adsorption of components with opposite charges. During desorption or elution, sample components are exchanged by ions present in the solution or buffer used to elute, or by a change in pH that alters the net charge of the molecule of interest.
Reverse phase adsorption chromatography involves separating the sample components according to their different polarities. Sample components are adsorbed through a resin composed of a silica matrix covered with an organic polymer by non-covalent bonding. The selective desorption of the components occurs afterwards by the elution with a non-polar solvent containing the eluent.
The separation techniques described above were utilized initially to separate relatively small hydrophobic or hydrophilic molecules. Their application to the purification of larger molecules, such as proteins, and specially complex proteins such as lipoproteins, nucleoproteins and glycoproteins, is more recent. Numerous publications illustrate the state of the art attained so far in protein separation.
See Soferet et al., “Handbook of Process Chromatography” (Academic Press Inc., San Diego, Calif., 1997); Olson, Ed., “Separation Technology” (Interpharm Press, Inc., Buffalo Grove, Ill., 1995); Franks, Ed., “Protein Biotechnology” (Human Press, Totowa, N.J., 1993); Deutscher, Ed., “Guide to Protein Purification, Methods in Enzymology”, Vol. 182, (Academic Press Inc. San Diego, Calif., 1991); Seetharam et al., Eds., “Purification and Analysis of Recombinant Proteins” (Marcel Dekker, Inc., New York, N.Y., 1991); Harria et al., Eds., “Protein Purification Applications” (Oxford University Press, Oxford, England, 1990); Brown, et al., Analytical Biochemistry, 99, 1–21, 1979; Harrison et al., “VDYAC TM Comprehensive Guide to Reverse Phase Materials for HPLC”, pp. 1-12 (The Sep/A/Ra/Tions Groups, Hesperia, Calif., 1984). The use of monoclonal antibodies raised against the protein of interest is another known method of protein recovery.
Several specific methods for recombinant EPO separation have been recently reported. One of these methods consists in protein purification by anionic exchange chromatography with selective protease elimination, followed by reverse phase chromatography and filtration. See U.S. Pat. No. 4,667,016 (to Lai et al.). This technique claims a yield of 16% EPO of unknown specific activity and purity.
Another method proposed for the separation of recombinant EPO consists in the application of reverse phase high pressure liquid chromatography (RP-HPLC) to a solution containing partially purified protein. See U.S. Pat. No. 4,667,195 (to Hewick et al.). This method has been found irreproducible in practice. Morever, the non-polar solvents commonly employed or recommended for protein and polypeptide separation by means of RP-HPLC, include reagents such as acetonitrile, difficult to remove from the protein of interest and potentially toxic for human beings. See Parsons, et al., Endocrinology, 114, 6, 2223–7 (1984). It should be noted, however, that ethanol and formic acid aqueous solutions for protein elution have also been used. See Takagaki, et al., Journal of Biological Chemistry, 5, 4, 1536–41 (1980).
Even though there is abundant information regarding the production of recombinant human EPO, a purification method yielding EPO adequate for its utilization in human beings has not yet been described. A suitable protein purification method should yield EPO over 99% pure and free of contaminants such as: aggregated material, b) degraded material, c) spurious proteins and d) proteases. A protein purity under 99% or the presence of any of the above mentioned contaminants might be toxic for human beings.
On the other hand, many of the methods proposed for EPO purification are not efficient when applied to industrial scale protein production. The RP-HPLC method employs expensive organic solvents, which increases purification costs. In addition, organic solvents are more difficult to handle and contaminant to the environment. Other purification methods proposed are irreproducible in practice or have a low yield.